Tailored Watson–Crick Pairing Partners for Xanthosine in RNA

The deamination of guanosine nucleobases results in the formation of xanthosine (X)-containing RNA. This process formally resembles adenine deamination (A-to-I editing). The biological significance and basic chemical consequences of X-modified RNA are poorly understood. In this study, we examine xanthosine interactions with tailored pyrimidine nucleobases. We synthesized RNAs that provide either the 2,4-diaminopyrimidine C-nucleoside kappa (K) or 2-iminocytidine (2imC) as an "ideal" tridendate Watson-Crick pairing partner. For K-modified RNA, we used a phosphoramidite previously introduced for RNA solid-phase synthesis. For 2imC-modified RNA, we developed access via synthetic 2-thiocytidine (2sC)-modified RNA, followed by osmium tetroxide (OsO₄)-ammonium chloride (NH₄Cl)-mediated transformation into 2imC-modified RNA. RNAs with K-X or 2imC-X base pairs indeed showed stable duplex formation with significantly higher melting temperatures than X-C or X-U containing RNAs and higher still than A-U or G-U containing RNA references. Furthermore, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallographic structure analysis confirmed the tridendate Watson-Crick pair geometry of K-X within an RNA double helix. Our findings suggest that K-X and 2imC-X pairs could be used in RNA decoding processes, thereby expanding the RNA genetic alphabet. However, their potential for use in biochemical and cellular applications remains to be seen.